The invention relates to the use of carbopyronine compounds of the general formula (I) as labeling groups in procedures for the detection of analytes, to novel carbopyronine compounds and to a process for the preparation of these compounds.
In chemical, medical and biological analysis, dyes are used as labeling or detection groups. In particular, fluorescent dyes have gained importance in recent years and displaced other often cost-intensive procedures, which use, for example, radioisotopes for labeling.
In particular in the field of DNA sequencing, fluorometric procedures have gained acceptance in recent years and almost completely replaced the procedures customary up till then, which use radioactive isotopes.
In spite of the availability of various fluorescent dyes, such as, for example, FITC (fluorescein isothiocyanate), FLUOS (fluorescein N-hydroxysuccinimide ester), rhodamine derivatives etc., it was previously not possible to solve the problems due to background fluorescence, honspecific binding phenomena and the need for cost-intensive measuring equipment in a satisfactory manner.
As a result of background fluorescence and nonspecific binding, the sensitivity and accuracy of the measurements is reduced. In addition, in the case of available fluorescent dyes the absorption maximum lies in regions which do not make possible the use of light sources which are less expensive and which can be of small dimensions, such as, for example, He/Ne lasers and laser diodes.
An object of the present invention was thus to make available fluorescent dyes which can be employed as labeling groups in procedures for the detection of analytes and at least partially avoid the disadvantages of the prior art.
This object has been achieved by the use of compounds of the general formula (I) 
as labeling groups in a procedure for the detection of an analyte, where
R1, R2, R3, R4, R5, R6 and R7 are in each case independently hydrogen, halogen, a hydroxyl, amino, sulfo or carboxyl or aldehyde group or a saturated or unsaturated, straight-chain, branched or cyclic hydrocarbon group having up to 20 C atoms, where the hydrocarbon groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, in particular phenyl, or/and heteroaryl radicals and optionally heteroatoms such as oxygen, sulfur or nitrogen atoms or/and two or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde, C1-C4-alkoxy or/and C1-C4-alkoxycarbonyl groups, or one or more of the radicals R1-R7, in each case with adjacent substituents, form a ring system which can contain one or more multiple bonds,
R8 and R8a in each case independently are a saturated or unsaturated, straight-chain, branched or cyclic hydrocarbon group having up to 20 carbon atoms, e.g. a C1-C6-alkyl group, in particular methyl, ethyl, propyl or/and butyl, or an aryl or heteroaryl group, in particular phenyl, which optionally contain heteroatoms such as oxygen, sulfur or nitrogen atoms or/and one or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, phospho, carboxyl, aldehyde, C1-C4-alkoxy or/and C1-C4-alkoxycarbonyl groups, or R8 and R8a can form a ring system,
R9, R10, R11 and R12 in each case independently are hydrogen or a saturated or unsaturated, straight-chain, branched or cyclic hydrocarbon group having up to 20 C atoms, e.g. polyether, phenyl, phenylalkyl having 1-3 C atoms in the chain, where the hydrocarbon groups can optionally contain heteroatoms such as oxygen, sulfur or nitrogen atoms or/and one or more substituents, preferably selected from halogens, hydroxyl, amino, sulfo, phospho, carboxyl, carbonyl, alkoxy or/and alkoxycarbonyl groups,
or one or more of the radicals R9-R12, in each case with adjacent substituents, form a ring system which can contain one or more multiple bonds,
where xe2x80x94N(R11)(R12) or/and xe2x95x90(R9)(R10) can be replaced by xe2x80x94OR9 or/and xe2x95x90O,
and X is optionally anions present for charge equalization.
The compounds of the general formula (I) can be employed as labeling groups in procedures for the qualitative or/and quantitative determination of an analyte. This determination can be carried out in aqueous liquids, e.g. samples of body fluids such as, for example, blood, serum, plasma or urine, wastewater samples or foodstuffs. The procedure can also be carried out as a wet test, e.g. in a cuvette, or as a dry test in an appropriate reagent carrier. The determination of the analyte can be carried out here by means of a single reaction or by means of a sequence of reactions. Surprisingly, the use of compounds of the general formula (I) showed very good results in chemical and in particular in medical and biological detection procedures for the determination of an analyte, especially in nucleic acid sequencing procedures and in protein analysis.
The compounds of the general formula (I) can be used in all chemical, medical and biological detection procedures known to the person skilled in the art in which fluorescent dyes are suitable as labeling groups. For this, the compounds of the general formula (I) are in general coupled covalently to a receptor which is specific for the analyte to be detected. This takes place using generally known procedures. The specific receptor can be any suitable compound or any suitable molecule, preferably it is a peptide, a polypeptide or a nucleic acid. The compounds or conjugates of these compounds can be used, for example, in nucleic acid, hybridization procedures, in particular for the sequencing of nucleic acids or immunochemical procedures. Procedures of this type are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor.
A further object of the present invention was to make available novel carbopyronine compounds which are suitable in particular for use as labeling groups in analyte detection procedures, can be prepared using simple and inexpensive processes, can be handled without problems and at least partially avoid the disadvantages of the prior art.
This object has been achieved by a compound of the general formula (I) 
where
R1-R12 and X have the meanings indicated above,
with the proviso that if R1-R3 and R5-R7 are hydrogen and R8, R8a and R9-R12 are methyl,
R4 is not hydrogen, methyl, isopropyl, phenyl, 2,6-dimethylphenyl or 2-isopropenylphenyl.
An advantage of the compounds (I) is that owing to an almost arbitrary substituent variation the properties of individual compounds, e.g. the spectroscopic properties, the position of the absorption and fluorescence maxima, the solubility properties, the fluorescence quantum yield and decay time, vary strongly and thus can be selected as desired. In this way, interferences with interfering substances in samples such as serum, blood or plasma etc. can be reduced or even avoided completely. The preparation of some compounds of the formula (I) can be carried out by processes known per se. Preferably, the synthesis is carried out, however, according to a novel process described below, which is particularly simple and inexpensive.
In a preferred class of the compounds (I), R6 is bridged with R11 or/and R7 with R12, R1 with R10 or/and R2 with R9 and form a ring system which can contain one or more multiple bonds. The ring system preferably contains one or more 5- or 6-membered rings.
R4 is preferably hydrogen, C1-C6-alkyl or a radical containing an aromatic ring system, e.g. a radical containing a carboxyl or/and halogen group, such as 2-carboxyphenyl, 2-carboxytetrachlorophenyl or pentafluorophenyl. R8 and R8a are preferably in each case independently methyl, ethyl or/and optionally substituted phenyl.
Examples of particularly preferred classes of compound are shown in the general formulae IVa to IVe: 
in which the dashed lines are optionally double bonds, in whose presence the radicals R bonded via a dashed line are absent,
R1, R3, R4, R5, R6, R7, R8, R8a, R9, R11, R12 and X are as defined above, and R, on each occurrence, can be identical or different and is defined as R1-R7 above.
The compounds preferably have a group capable of covalent coupling, e.g. xe2x80x94COOH, xe2x80x94NH2, xe2x80x94OH or/and xe2x80x94SH. By means of this coupling group, the compound can be coupled to a carrier or/and to a biomolecule according to known methods. The carrier can consist of any material which is suitable, in particular for detection procedures, e.g. of porous glass, plastics, ion-exchange resins, dextrans, cellulose, cellulose derivatives or/and hydrophilic polymers. The biomolecules are preferably selected from peptides, polypeptides, nucleotides, nucleosides, nucleic acids, nucleic acid analogs or/and haptens.
Surprisingly, the absorption maxima and the fluorescence quantum yield are not significantly changed by coupling of the compounds according to the invention to the abovementioned carriers and biomolecules.
Actual examples of compounds according to the invention are shown in table 1 below.
A further object of the present invention consisted in making available a preparation process for carbopyronine compounds which can be carried out in a simple, environmentally compatible and inexpensive manner and which at least partially avoids the disadvantages of the known processes for the preparation of carbopyronines.
This object was achieved according to the invention by a process for the preparation of compounds of the general formula (I) 
where R1-R12 and X have the meanings indicated in claim 1, characterized in that a compound of the general formula (II) 
in which R5, R6, R7, R8, R8a, R11 and R12 are as defined above, or the dehydration product of II is reacted with a compound of the general formula III 
in which R1-R4, R9 and R10 are as defined above and Y is a halogen, in particular bromine, a hydroxyl or thio group, in a suitable solvent, under acidic conditions and in the presence of a catalyst and the compound formed by ring closure between the compounds II or their dehydration product and III is reacted by oxidation to give the structure I.
In the process, it is possible to use all suitable solvents which are compatible with the starting materials, the products and the catalyst, preferably boron trichloride. The solvent is preferably a nonpolar solvent, in particular methylene chloride, 1,2-dichloroethane or chloroform.
The acids employed can be customary acids. The acid is preferably an inorganic acid such as sulfuric acid, phosphoric acid or polyphosphoric acid.
The oxidants used can likewise be customary oxidants. The oxidant tetrabutylammonium(meta)periodate is preferred.
It is particularly advantageous that the process can be carried out without isolation of intermediates. This leads to a reduction in the expenditure of time, labor and material.